Process for preparing 24-fluoro-25-hydroxycholecalciferol

ABSTRACT

The invention provides a new derivative of vitamin D 3 , 24-fluoro-25-hydroxycholecalciferol. 
     The compound is characterized by vitamin D-like activity in its ability to increase intestinal calcium transport, increase serum calcium and inorganic phosphorous concentration and to prevent the development of rickets. It would find ready application as a substitute for vitamin D 3  and in the treatment of disease states evincing calcium-phosphorous imbalance and which are nonresponsive to vitamin D 3  therapy.

The invention described herein was made in the course of work under agrant or award from the Department of Health and Human Services, andU.S. Japan Cooperative Grant INT-76-05793 and IPA No. 0001 awarded bythe National Science Foundation.

TECHNICAL FIELD

This invention relates to a compound which is characterized by vitaminD-like activity.

More specifically this invention relates to a method for preparing aderivative of vitamin D₃ and the new and novel intermediates in suchprocess.

Vitamin D₃ is a well-known agent for the control of calcium andphosphorous homeostasis. In the normal animal or human this compound isknown to stimulate intestinal calcium transport and bone-calciummobilization and is effective in preventing rickets.

It is also now well known that to be effective vitamin D₃ must beconverted in vivo to its hydroxylated forms. For example, the vitamin D₃is first hydroxylated in the liver to form 25-hydroxy vitamin D₃ and isfurther hydroxylated in the kidney to produce 1α,25-dihydroxyvitamin D₃or 24,25-dihydroxy vitamin D₃. The 1α-hydroxylated form of the vitaminis generally considered to be the physiologically active or hormonalform of the vitamin and to be responsible for what are termed thevitamin D-like activities, such as increasing intestinal absorption ofcalcium and phosphate, mobilizing bone mineral, and retaining calcium inthe kidneys.

BACKGROUND ART

References to various of vitamin D derivatives are extant in the patentand other literature. See, for example, U.S. Pat. Nos. 3,565,924directed to 25-hydroxycholecalciferol; 3,697,559 directed to1,25-dihydroxycholecalciferol; 3,741,996 directed to1α-hydroxycholecalciferol; 3,907,843 directed to1α-hydroxyergocalciferol; 3,715,374 directed to24,25-dihydroxycholecalciferol; 3,739,001 directed to25,26-dihydroxycholecalciferol; 3,786,062 directed to22-dehydro-25-hydroxycholecalciferol; 3,847,955 directed to1,24,25-trihydroxycholecalciferol, 3,906,014 directed to3-deoxy-1α-hydroxycholecalciferol; 4,069,321 directed to the preparationof various side chain fluorinated vitamin D₃ derivatives and side chainfluorinated dihydrotachysterol₃ analogs; 4,188,345 directed to a processfor preparing fluorinated vitamin D compounds; 4,196,133 directed to24,24-difluoro-25-hydroxycholecalciferol.

DISCLOSURE OF INVENTION

A new method for preparing 24-fluoro-25-hydroxycholecalciferol(24-fluoro-25-hydroxyvitamin D₃ or 24-F-25-OH-D₃) has now been found bywhich said compound can be readily prepared. 24-F-25-OH-D₃ expressesexcellent vitamin D-like activity as measured by its ability tostimulate calcium transport in intestine and its ability to increaseserum calcium concentration. Such compound, therefore, could serve as asubstitute for vitamin D in its various metabolic bone diseases such asostenmalacia, osteodystrophy and hypoparathyroidism.

BEST MODE FOR CARRYING OUT THE INVENTION

24-F-25-OH-D₃ was synthesized in accordance with the followingdescription and abbreviated schematic: ##STR1##

SYNTHESIS of 24-FLUORO-25-HYDROXYCHOLECALCIFEROL

For the synthesis of 24F-25-hydroxyvitamin D₃ 11, cholenic acid issuccessively treated with dihydropyran-p-TsOH, LiAlH₄, andp-TsCl-pyridine to afford the tosylate 1, which in turn is reacted withKCN/18-crown-6 in dimethylformamide at 70° C. for 3 hr. to give thecyanide 2 (48% yield, mp 142-143). Hydrolysis of 2 with KOH in aqueousethanol (140°, 48 hr.) yielded the carboxylic acid 3 (63% yield, mp.171°-172° C.), which on treatment with CH₂ N₂ gives the methyl ester 4(79% yield, mp 159°-161°). The enolate of 4 (generated by treatment withlithium dicyclohexylamide at -78° C.) was treated with iodine in THF at-78° C. to yield the idodide 5. Substitution of iodine of 5 withhydroxyl was accomplished by reaction with CF₃ COOAg-Ag₂ O in CH₃CN-ethyl ether (20° C., 16 hr.). Reesterification with CH₂ N₂ gave thehydroxy ester 6 (mp 114°-117° C., δ4.16 (1H, m, C-24), m/e 418 (M-84))in 63% overall yield from 4. (The 3,24-dibenzoyl ester derived from 6showed a twin peak on high pressure liquid chromatography (HPLC)(Zorbax-SIL, 10% CH₂ Cl₂ in n-hexane), indicating that 6 is a 1:1epimeric mixture at C-24 position. This was further corroborated bytransformation of 6 into a 1:1 mixture of (24R)- and(24S)-3,24-dibenzoyloxycholest-5-ene-25-ol trimethylsilyl ether whichwas cochromatographed on HPLC with authentic samples (M. Seki, N.Koizumi, M. Morisaki and N. Ikekawa, Tetrahedron Letters, 15 (1973)).)

The tosylate 7 derived from 6 was treated with KF/18-crown-6 indimethylformamide (70° C., 15 hr.) to give the fluoride 8, which onhydrolysis (p-TsOH in methanol-CH₂ Cl₂, 0°, 4 hr.) affords the fluoroester 9 (73% yield, mp 104°-105° C. δ4.86 (1H, dm, J_(ht) =48 Hz, C-24),m/e 420 (M⁺)). Treatment of 9, which, if desired can be reprotected atC₃ by acylation (e.g. acetylation, benzoylation) by known procedures,with an excess of CH₃ MgI in ethyl ether (20° C., 20 min) followed byacetylation furnished in 80% yield, the fluorohydrin 10 (mp 153°-154°C., δ4.14 (1H, dm, J_(Hf) =48 Hz, C-24), 1.20 (6H, S, C-26,27), m/e 402(M-60)). Conversion of 10 to the corresponding vitamin D₃ 11 was carriedout essentially as described for24,24-difluoro-25-hydroxycholecalciferol in U.S. Pat. No. 4,196,133,issued Apr. 1, 1980 and in Y. Kobayashi, T. Taguchi, T. Terada, J.Oshida, M. Morisaki and N. Ikekawa. Synthesis of 24,24-difluoro and24-fluoro-25-hydroxyvitamin D₃. Tetrahedron Letters, No. 22, pp. 2020(1979).

Compound 11 showed the expected spectral properties (λ_(max) 263, nm,λ_(min) 228, nm, m/e 418 (M⁺), 402 (M-15), 400 (M-18), 385 (M-15-18),359, 271, 253, 136, 118).

BIOLOGICAL ACTIVITY

Male weanling rats were purchased from Holtzman Company, Madison, Wis.,were housed in hanging wire cages and were fed ad libitum water and thelow calcium vitamin D-deficient diet as described by Suda et al. (J.Nutrition 100, 1049, 1979) for three weeks prior to their use in thefollowing assays.

INTESTINAL CALCIUM TRANSPORT

Rats that had been fed the low-calcium, vitamin D-deficient dietdescribed above were divided into three groups of 5 rats each. The ratsin each group were then respectively given a single dose of 650 pmole ofeither 24-F-25-OH-D₃ or 25-OH-D₃ dissolved in 0.1 ml 95% ethanolintrajugularly 23 hours prior to sacrifice. Rats in the control groupswere given the ethanol vehicle only in the same manner. The rats in thegroups were killed by decapitation and their blood was collected for themeasurement of serum calcium concentration. Their duodena were used tomeasure intestinal calcium transport activity in accordance with thetechnique of Martin and DeLuca (Am. J. Physiol. 216, 1351, 1969) withthe results shown in Table I, second column.

SERUM CALCIUM CONCENTRATION

The blood collected as described above was centrifuged to obtain theserum. One-tenth ml of serum was mixed with 1.9 ml of 0.1% lathanumchloride solution and the calcium concentration was measured with anatomic absorption spectrophotometer (Perkin-Elmer Model HO-214). Resultsare shown in Table 1, third column.

It is evident from Table I, that 24-fluoro-25-hydroxyvitamin D₃ exhibitspronounced vitamin D-like activity as evidenced by the increase ofintestinal calcium transport and increase in the concentration of serumcalcium and appears to be wholly as effective in this regard as25-hydroxyvitamin D₃ (see U.S. Pat. No. 3,565,924).

                  TABLE I                                                         ______________________________________                                        Intestinal Calcium Transport and Increase in Serum Calcium                    Concentration in Response to a Single Dose of 650 pmole 24-F-                 25-OH--D.sub.3 or 25-OH--D.sub.3 Given 24 hrs. Prior to Sacrifice.                        Intestinal Calcium                                                                              Serum                                           Compound    Transport         Calcium                                         Given       .sup.45 Ca serosal/.sup.45 Ca mucosal                                                           mg/100 ml                                       ______________________________________                                        ethanol (control)                                                                          1.5 ± 0.4*.sup.a                                                                            5.0 ± 1.1.sup.d                              24-F-25-OH--D.sub.3                                                                       3.7 ± 0.5.sup.b                                                                              8.4 ± 0.4.sup.e                              25-OH--D.sub.3                                                                            4.5 ± 0.8.sup.c                                                                              7.7 ± 0.4.sup.f                              ______________________________________                                         *Standard deviation of the mean. Significance of difference:                  .sup.b & .sup.c from .sup.a p<0.001                                           .sup.b from .sup.c N.S.                                                       .sup.e from .sup.d p<0.001                                                    .sup.f from .sup.d p<0.005                                                    .sup.e from .sup.f p<0.05                                                

What is claimed is:
 1. 24-fluoro-25-hydroxycholecalciferol. 2.24-fluoro-25-hydroxy previtamin D₃.
 3. Compounds having the formula##STR2## where each of R and R₁ is hydrogen or acetyl.
 4. Compoundshaving the formula ##STR3## where each of R and R₁ is acetyl orhydrogen.
 5. Compounds having the formula ##STR4## where R istetrahydropyranyl, hydrogen, or acyl.
 6. A method for preparing24-fluoro-25-hydroxycholecalciferol which comprises:converting3-tetrahydropyranyl homocholenic acid ester to its enolate by treatmentwith lithium dicyclohexylamide and treating the enolate with iodinewhereby 3-tetrahydropyranyl-24-iodo-homocholenic acid ester is obtainedconverting said 24-iodo-homocolenic acid ester to3-tetrahydropyranyloxy-24-hydroxy homocholenic acid by treatment withCF₃ COORAg--Ag₂ O reesterifying said 3-tetrahydropyranyloxy-24-hydroxyhomocholenic acid with diazomethane to obtain3-tetrahydropyranyloxy-24-hydroxy homocholenic acid methyl ester andconverting said ester to 3-tetrahydropyranyloxy-24-tosyloxy-homocholenicacid methyl ester treating said tosylate with potassium fluoride toobtain 3-tetrahydropyranyloxy-24-fluoro-homocholenic acid methyl esterhydrolyzing said 3-tetrahydropyranyloxy-24-fluoro-homocholenic acidmethyl ester to obtain 24-fluoro-homocholenic acid methyl ester treatingsaid ester with a Grignard reagent followed by acetylation to produce24-fluoro-25-hydroxy-cholesterol 3-acetate allylically brominating saidcholesterol acetate followed by dehydrobromination and separating3-acetoxy-24-fluoro-25-hydroxy-5,7-cholestadiene saponifying said dieneand exposing the resulting 3,25-dihydroxy-24-fluoro-5,7-cholestadiene toactinic radiation whereby 24-fluoro-25-hydroxy previtamin D₃ is obtainedthermally isomerizing said previtamin and recovering24-fluoro-25-hydroxy-cholecalciferol.